Fast, precise, and accurate

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1 B i op r o c e s s Technical Development and Qualification of a Generic IgG Quantification Assay Using Surface Plasmon Resonance Stefan Winheim, Anton Walser, and Matthias Kania Fast, precise, and accurate quantification technologies are indispensable for efficient process development in applications such as IgG production in a GXP environment. Based on surface plasmon resonance (SPR) technology, the Biacore C system from GE Healthcare ( is an alternative technology for IgG quantification that has benefits over traditional methods. Assay development is simplified and accelerated due to real-time detection. Assay hands-on time is reduced, and sample throughput can be increased using automation and efficient data evaluation with regulatory-compliant software. We developed a generic platform for higg quantification using the Biacore C instrument. Materials And Methods Immobilization of Mouse Anti-hIgG: Mouse anti-higg (antihuman Fc) was covalently bound as a ligand by Product Focus: IgG antibodies Process Focus: Process development Wh o Sh o u l d Re a d: QA/QC, p r o d u c t a n d p r o c e s s development, a n a l y t i c a l, and manufacturing personnel Ke y w o r d s : Co n c e n t r a t i o n, assay development, m e t h o d qualification, validation, robustness Le v e l: Intermediate Biacore C instrument ge healthcare ( means of primary amino groups on the carboxy-methylated dextran molecules of a sensorchip surface. We performed all our measurements using research-grade Biacore CM5 chips. Mouse anti-higg was immobilized using amino coupling chemistry and 1 mm acetate buffers at different ph conditions. Concentration Measurement: We selected the concentration analysis wizard from the Biacore software to perform our concentration analysis, enabling optimal settings. Samples and standards were injected at a flow rate of µl/min and a contact time of 1 seconds over the immobilized flow cell. For data evaluation, we calculated the signal differences (shown as resonance units) by setting report points 1 seconds before starting the injection of analyte or standard and 3 seconds after the initial injection. After each signal generation, the sensorchip was regenerated to dissociate the analyte ligand binding and to allow a new analyte injection. Both the response to analyte injection and regeneration were displayed in one sensorgram. To evaluate the working range of the assay, we performed a preliminary standard curve comprising a range of concentrations of higg (Ab) and evaluated the curve using a fourparameter equation. Comparison with the standard curve allowed quantification of unknown higg in samples. We examined the accuracy of this analytical method through quantification of five different cell culture samples by comparing their Ab b r e v iati o n s Ab: antibody CV: coefficient of variation EDC: 1-ethyl-3(3-dimethylaminopropyl) carbodiimide ELISA: enzyme-linked immunosorbent assay higg: human immunoglobulin G HPLC: high-performance liquid chromatography ICH: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use LOD: limit of detection LOQ: limit of quantitation NHS: N-hydroxysuccinimid PBS: phosphate buffered saline RU: resonance units SPR: surface plasmon resonance 3 BioProcess International Jun e 9

2 Figure 1: Preconcentration of ligand in the dextran matrix; 1 seconds injection of mouse anti-higg (Invitrogen) diluted to 5 µg/ml in acetate buffers of different ph values Signal ( 1, RU) ph Value data with the results of a validated protein A-HPLC assay and the results of a commercially available IgG- ELISA from Bethyl Laboratories (catalog number E8-1, www. bethyl.com). To check the precision of our assay, we quantified one cell culture sample containing higg in four different dilutions covering the whole working range. The test was performed on five different days using five different flow cells to determine the assay s intermediate precision. To test the robustness of our assay with regard to storage conditions, we immobilized the ligand (mouse antihigg) on flow cells of two different sensorchips. One sensorchip was stored under dry conditions in a bluecap vial at 8 C, whereas the other was stored in a blue-cap vial filled with Biacore running buffer HBS-EP at 8 C. Over one month, we performed several standard curves and compared their responses. During purification of antibodies on protein A columns, small amounts of capture molecules often leach into the eluate along with a product antibody. The binding of protein A to it, therefore, may affect the accurate measurement of a product antibody. To address this, we tested the competitive influence of protein A with regard to the accuracy of antibody quantification by preparing solutions of 15 ng/ml higg (Ab), spiking them with different concentrations of protein A, and analyzing them four times on one Figure : Regeneration scouting used 1 mm glycine-hcl solutions of different ph values; influence to the baseline (green) and to the signal responses (blue) injecting 1, ng/ml higg (Ab); cycles 1 7 = ph 3.; cycles 8 1 = ph.5; cycles 15 1 = ph.; cycles 8 = ph 1.5. Signal (RU) flow cell. Kinetic Test Evaluating the Generic IgG Platform: To check the binding characteristics of different antibodies to the immobilized ligand, we performed kinetic analyses. We determined association and dissociation rates by injecting each analyte in different concentrations over the immobilized sensorchip. To normalize buffer effects, each analyte was sequentially injected over the detection flow cell and then over the reference flow cell. The signals were subtracted from each other during the data evaluation stage. We determined the association rates (k a ), dissociation rates (k d ), and dissociation constants (K D ) in three independent assays by displaying the sensorgrams in overlay plots and fitting them on a 1:1-Langmuir model using BIAevaluation software from GE Healthcare. Re s u lt s Ligand Immobilization and Regeneration: Mouse anti-higg from Invitrogen ( was covalently immobilized as a ligand. A high level of immobilized ligand is important for sensitive concentration assays. We diluted this ligand in acetate buffers with different ph values and injected it over a nonactivated flow cell to determine Figure 3: Standard curve with the concentrations of 1, ng/ml higg (Ab), 5, ng/ml higg (Ab),,5 ng/ml higg (Ab), 1,5 ng/ml higg (Ab), 5 ng/ml higg (Ab), 31.5 ng/ml higg (Ab), 15.3 ng/ml higg (Ab), 78.1 ng/ml higg (Ab), 39.1 ng/ml higg (Ab), and ng/ml higg (Ab) Signal ( 1 RU) , 3, 3, 3,38 3,3 3,3 3,3 3,3 3,8 3, Cycle Baseline (RU) 8 1 higg ( 1, ng/ml) the best electrostatic concentration in the dextran matrix. The best electrostatic concentration of the ligand came with 1 mm acetate buffer at ph 5. as a dilution buffer (Figure 1). Using this buffer for the immobilization procedure, we obtained an average immobilization level of ~11, resonance units (RU) (data not shown). After each sample injection, the sensorchip surface was regenerated to dissociate the analyte from the ligand and prepare the flow cell for a new sample injection. We screened regeneration solutions to find a solution that fully dissociates the analyte ligand binding without affecting the ligand s binding activity. Scouting glycine-hcl solutions at the ph values of 3.,.5,., and BioProcess International Jun e 9

3 Table 1: Intermediate precision; each concentration was measured on different flow cells and on different days (interflow cell precision; n = 5). Mean (ng/ml) STDEV (ng/ml) CV (%) Confidence Interval (µg/ml)* * P = 95% 9, , Table : Average association and dissociation rates are listed with the dissociation constants of different antibodies to the immobilized ligand (n = 3). Antibody k a (1/Ms) (CV) k d (1/s) (CV) K D (nm) (CV) higg1 (Ab5) 1. 1 (7.3%) (1.7%) 3 (1.%) higg (Ab). 1 (.1%). 1 3 (13.3%) 19 (18.1%) higg3 (Ab7) (15.%) (3.%) 7 (5.8%) higg (Ab8).5 1 (1.9%). 1 3 (17.%) (5.1%) higg1 (Ab3).1 1 (.1%) (1.3%) 1 (1.1%) higg1 (Ab) (.1%) (11.7%) (13.5%) were screened for seven cycles, respectively (Figure ). In each cycle, 1, ng/ml of higg was injected for 1 seconds and then regenerated by injections of regeneration solution for 3 seconds. A stable baseline combined with a high and stable signal was obtained using 1 mm glycine-hcl at ph.5. An immobilized flow cell could be used for ~8 sample cycles under those conditions (data not shown). Working Range, Limit of Detection (LOD), and Limit of Quantitation (LOQ): To evaluate the working range of the assay, we generated a standard curve using purified higg (Ab). The results in Figure 3 show the standard curve ranging , ng/ml in this Biacore assay. We set the working range of the assay from 15.1 to 1, ng/ml, which was confirmed by the experiments described below. Furthermore, we calculated the LOD by a signal-to-noise ratio of 3:1, which corresponds to an analyte concentration of 5 ng/ml higg. We determined the LOQ using a signal-to-noise ratio of 1:1, which corresponds to an analyte concentration of 1 ng/ml. Specificity, Accuracy, and Precision: The minimum dilution of upstream and downstream samples must be determined for each step individually. In our test, we analyzed analyte-free cell culture medium in different dilutions to evaluate nonspecific signals (Figure ). For comparison, we injected HBS-EP buffer eight times, which resulted in an average signal of.5 ±. RU. As Figure shows, cell culture samples should be diluted at least 1: to prevent unspecific binding. From a dilution of 1:, the corresponding resonance units (~ RU) were in the range of the blank (.5 ±. RU). Therefore, in cell culture supernatants the LOD was ng/ml and the LOQ was ng/ml. We examined the accuracy of the method by quantifying five different cell culture samples and comparing the results with those of a validated protein A-HPLC assay and a commercially available IgG-ELISA. The detected IgG concentrations compared well with both the validated protein A-HPLC assay and the ELISA. With the HPLC results set at 1%, we obtained recovery rates of 9 1% with the Biacore assay. We checked the intermediate precision of our Biacore assay by quantifying one higg-containing cell culture supernatant in four different dilutions on five different days and on five different flow cells (Table 1). The results of our study demonstrated precise results over the examined working range (1 1, ng/ml) with a maximum coefficient of variation (CV) of 5.7%. However, for practical reasons, we set the working range at , ng/ml. Robustness: We performed robustness tests to evaluate chip storage conditions and process-related impurities (Figures and 7). Ligand activity decreased ~9% within one month when the sensorchip was stored under dry conditions (Figure ). By contrast, when the sensorchip was stored in HBS-EP over a month, only a ~5% loss of ligand activity could be detected (Figure 7). We also tested the competitive influence of protein A on the accuracy of antibody quantification and found that protein A concentrations <1 ng/ml had no effect on the quantitation of 15 ng/ml higg (Figure 8). Affinity Dependence of a Generic Assay: Generic IgG concentration assays should be independent of the affinity between analytes and the immobilized ligand so that different immunoglobulins can be detected on one standard curve. In our tests, we checked the affinity dependence of the assay and performed kinetic measurements to compare the binding behavior with recovery rates. We quantified the recovery of different higgs on one standard curve to check whether the developed assay could be used as a generic platform. We tested four wild-type antibodies as analytes (Ab5,, 7, and 8), representing the four higg subclasses higg1 to higg. Two monoclonal higg1 (Ab3 and Ab) antibodies were also tested. As a positive control, higg (Ab) was quantified on the standard curve, which was performed using higg (Ab). Each analyte was diluted with HBS-EP running buffer to a final protein concentration of 1 µg/ml and injected five times. We calculated the recovery rates based on a target concentration of 1 µg/ml (Figure 9). Significant under- and overestimation could be shown by quantifying different antibody subtypes on one standard curve. A recovery rate of ~1% was reached only when analyte and standard material were identical (Ab). We determined the concentrations of three wild-type antibodies higg1 (Ab5), higg (Ab), and higg (Ab8) with recovery rates of ~85%, whereas the wild-type antibody higg3 (Ab7)

4 Figure : Analyte-free cell culture supernatant was analyzed in different dilutions to check minimum required dilution. Signal (RU) , was detected with a recovery rate of ~73%. The recovery rates of both monoclonal higg1 antibodies were ~15% and 3%, respectively. However, we obtained high precision (CV <%) for each antibody quantification. To analyze the different analyteligand binding behavior, we subsequently performed affinity studies. By contrast with concentration assays, only small amounts of ligand should be immobilized for kinetic assays. For this purpose, we performed our assay with an immobilization level of RU. The results of the kinetic studies (Table ) correlated very well with the recovery rates of the standard dependent measurements (Figure 9). The Dilution Factor Figure 5: Precision and accuracy of Biacore assay, a protein A HPLC assay, and ELISA were compared. HIgG concentrations in cell culture supernatants were detected (n = ). Purified higg (Ab) was used as the standard material. higg (µg/ml) ELISA Biacore HPLC Clone 1 Clone Clone 3 Clone Clone 5 monoclonal higg1 with the highest recovery rates (Ab3 and Ab) also showed the highest association rates (k a ) of.1 1 and M -1 s -1. Comparable recovery rates of the three wild-type antibodies (Ab5,, and 8) were also reflected in comparable association rates from 1 1 to.5 1 M -1 s -1. The wild-type antibody higg3 (Ab7) showed both the lowest association rate ( M -1 s -1 ) and the lowest recovery rate (73%). Di s c u s s i o n To optimize the sensitivity of an immunoassay, high amounts of ligand must be immobilized to provide many binding sites for the ligand analyte interaction because the signal of a Biacore concentration assay directly depends on the amount of bound analyte (1). Under the elicited conditions (5 µg/ml anti-higg in 1 mm acetate at ph 5.), we obtained an average immobilization level of 11, RU. This corresponds to a protein concentration of ~11 ng/mm (). The common immobilization level for proteins of MW 5 15 kda is between 7, and, RU (3). Furthermore, complete regeneration of the analyte from the sensorchip surface is essential for an accurate and precise assay to provide the same binding capacity for each analyte injection (3). Besides the complete removal of the analyte, the conservation of ligand binding activity must also be considered. Incomplete regeneration or loss of binding activity from the surface will affect the performance of an assay and shorten the lifetime of the sensorchip (3). You can determine the quality of regeneration by comparing baseline signals from cycle to cycle and examine conservation of ligand binding activity by comparing analyte signals of repeated injections with identical analyte concentrations (Figure ). Glycine-HCl, ph 3., did not fully regenerate our sensorchip (shown by the increasing baseline signal). Evidently, harsher conditions at ph. influenced the ligand binding activity because the analyte signals obtained decreased from cycle to cycle. At ph 1.5 the ligand was clearly denaturized, (shown by strongly decreasing signals and an increasing baseline). The most successful regeneration was achieved by injecting glycine-hcl at ph.5, for 3 seconds. Under such conditions, immobilized flow cells could be used for 8 cycles. To elicit the working range, we generated a standard curve (39.1 1, ng/ml) and evaluated both LOD and LOQ by determining signal-to-noise ratio. After evaluation of the results obtained during accuracy and precision studies, we set the working range at , ng/ml. With about two log ranges, this assay is comparable with published Biacore concentration BioProcess International Jun e 9

5 Figure : Standard curves were performed over a period of one month on a dry-stored sensorchip (Ab1). Signal ( 1 RU) higg ( 1, ng/ml) 8 1 Day Day 1 Day Day 3 Day 5 Day Day 7 Day 1 Day 8 Figure 7: Standard curves were performed over a period of one month on a sensorchip stored in HBS-EP running buffer (Ab1). Signal ( 1 RU) higg ( 1, ng/ml) Day Day 1 Day Day 3 Day 5 Day Day 7 Day 1 Day 8 assays ( ). Compared with protein A-HPLC (working range 1 1 µg/ ml), the Biacore C method was more sensitive. The ELISA was the most sensitive method but also the least precise, with a working range of ng/ml. The Biacore assay described here can detect low titers with high precision. Therefore, this method can be used from the start of cell line development through product purification to release of a final product. By contrast, only higher higg concentrations can be measured with the traditional protein A-HPLC. Interflow cell measurements on different days covering the examined working range showed that the method provides good intermediate precision with CVs <%. Comparable Biacore antibody assays have CVs of % (5). The superior accuracy of the Biacore assay can be shown by quantifying different harvest samples and comparing the results with a validated protein A-HPLC and a commercial higg-elisa. With recovery rates of 9 1% compared with the HPLC, our assay is more accurate than other Biacore assays, which have recovery rates of 9 1% (). The recovery rates of the commercial ELISA were 9 11% compared with the HPLC and were thus less accurate than our Biacore assay. Because of the range of applications for surface plasmon resonance, an immobilized sensorchip must be stored outside the instrument. In a docked sensorchip, low buffer flow conserves ligand activity. Our testing of storage conditions for the sensor chip outside the Biacore C instrument showed that ligand activity could be best conserved in HBS-EP at 8 C for at least a month. Additionally, we tested the robustness of our assay with regard to process-related protein A impurity. Affinity chromatography in protein A columns is a common method in biotechnological processes for the purification of monoclonal antibodies following fermentation. Because of the high specificity of protein A constructs to the Fc-region of antibodies, a product purity of >98% can be obtained in a one-step process (7, 8). Usually, small amounts of leached protein A (up to 5 ng/ml) remain in the eluate of protein A columns (, 8). We show here that protein A concentrations up to 1 ng/ml did not influence higg quantification. Generally, IgG concentrations in protein A eluates are in the milligram range, so small amounts of protein A would not Figure 8: Competitive influence of protein A to the quantitation of 15 ng/ml higg (n = ) 15 1 Figure 9: Quantitation of different higgs on one standard curve (n = 5); each antibody was diluted to 1 µg/ml. The standard curve was prepared using higg (Ab). ng/ml 35 higg (n = ) 3 Recovery (%) Recovery (%) Protein A (ng/ml) 5 Ab3 Ab Ab5 Ab Ab7 Ab8 Ab higg1 higg higg3 higg Jun e 9 BioProcess International 1

6 significantly influence the quantitation. Apart from concentration, immunological methods also can be used to determine the biological activity of analytes, which is an advantage of surface plasmon resonance over analytical chromatography. In an HPLC assay, an immobilized ligand must bind to an epitope existing only in the active conformation of the analyte, and therefore inactive molecules are not bound (9 11). In Biacore assays, the total analyte amount and the amount of active molecules can be determined (1). With the protein A HPLC assay, only the total amount of protein can be determined. Therefore, HPLC assays must be combined with additional assays for activity determination. A generic assay that can quantify different higg subtypes on one standard curve is both time- and cost-saving. To produce accurate measurements, each analyte must bind the ligand with the same velocity to generate comparable signals during a defined injection time. The binding affinity between two molecules can be amplified by the substitution of one amino acid in the binding region of a molecule (13). Quantifying different antibodies on one standard curve showed that precise (CVs <%) but inaccurate results were obtained. Good recovery rates were reached only when the standard material and analyte were identical. This fact must be considered when a generic IgG assay is used in a biopharmaceutical environment. An Automated Alternative We developed and qualified a Biacore concentration assay for quantification of higg. The intermediate precision of <% and 9 1% recovery rates enabled precise and accurate quantification of higg in cell culture supernatants The elicited working range was , ng/ml higg. During robustness studies, we tested the effects of storage conditions and contaminations. Immobilized sensorchips always should be stored in HBS-EP running buffer to conserve ligand activity for several weeks. Furthermore, we have shown that protein A contaminations 1 ng/ml do not affect higg quantification in protein A eluates. Different immunoglobulins were quantified precisely but with an unacceptable level of accuracy on one standard curve. Due to affinity dependence, high accuracy was obtained only when the standard and analyte were identical. Kinetic studies proved that different immunoglobulins bound the ligand with varying association rates. Compared with other regulatorycompliant analytical methods, surface plasmon resonance has several favorable characteristics. Our Biacore assay is more precise and accurate than a commercially available IgG-ELISA, and hands-on time is reduced by its high degree of automation. Concentration measurements with a protein A-HPLC assay are less sensitive (µg range) than with the Biacore assay (ng range). Therefore, the latter can be used to analyze low-concentration samples, such as those occurring during early stages of cell culture processing. In addition, HPLC can detect only the total protein amount, not the biological activity of an analyte. Concentration measurements using analytical chromatography must be combined with activity assays to determine the concentration of active protein. By contrast, an appropriate Biacore assay can determine the total amount and the active amount of analyte in the same assay. Re fe re nces 1 Biacore Sensor Surface Handbook. Biacore AB (GE Healthcare): 3. Stenberg E, et al. Quantitative Determination of Surface Concentration of Protein with Surface Plasmon Resonance Using Radiolabeled Proteins. J. Colloid. Interface Sci. 1991: Biacore Concentration Analysis Handbook. Biacore AB (GE Healthcare): 1. Application Note 38: Rapid Development of a GMP-Compliant Biacore Assay for the Determination of Antibody Concentration. Biacore AB (GE Healthcare): 3. 5 Mason S, et al. Validation of the BIACORE 3 Platform for Detection of Antibodies Against Erythropoietic Agents in Human Serum Samples. Curr. Med. Res. Opin. 19(7) 3: Abraham R, Quimby M. Validation of a Biacore C Concentration Assay. BiaJournal 3, 3: Follmann D, Fahrner RL. Factorial Screening of Antibody Purification Processes Using Three Chromatographic Steps without Protein A. J. Chromatogr. A 1, : Fahrner RL, et al. Industrial Purification of Pharmaceutical Antibodies: Development, Operation and Validation of Chromatography Processes. Biotechnol. Gent. Eng. Rev. 18, 1: Wendler J, et al. Application of an SPR- Based Receptor Assay for the Determination of Biologically Active Recombinant Bone Morphogenetic Protein-. Anal. Bioanal. Chem. 381(5) 5: Zeder-Lutz G, Benito A, Van Regenmortel MH. Active Concentration Measurements of Recombinant Biomolecules Using Biosensor Technology. J. Mol. Recognit. 1(5) 1999: Sigmundsson K, et al. Determination of Active Concentrations and Association and Dissociation Rate Constants of Interacting Biomolecules: An Analytical Solution to the Theory for Kinetic and Mass Transport Limitations in Biosensor Technology and its Experimental Verification. Biochemistry 1() : Technical Note 3: Label-Free Protein Interaction Analysis in Real-Time Using Surface Plasmon Resonance. Biacore AB (GE Healthcare):. 13 Braden BC, et al. Anatomy of an Antibody Molecule: Structure, Kinetics, Thermodynamics and Mutational Studies of the Antilysozyme Antibody D1.3. Immunol. Rev. 13, 1998: For Further Reading ICH Harmonised Tripartite Guideline: Validation of Analytical Procedures: Text and Methodology Q(R1). Current Step Version, Parent Guideline 7 October 199 (Complementary Guideline on Methodology dated November 199, incorporated in November 5); MEDIA17.pdf (Link:.5.8). c Corresponding author Stefan Winheim is a scientist in quality control, Anton Walser is quality control team leader, and Matthias Kania is project leader of API production at Rentschler Biotechnologie GmbH in Mittelstraße 18, 8871 Laupheim, Germany; 9-739/71-, fax 9-739/71-3; stefan. winheim@rentschler.de; BioProcess International June 9